Imaging techniques gives insight to what happens in aged eggs

Currently, more and more women delay having children because of pursuing higher educational and career aspirations, as well as changing cultural norms. Unfortunately their eggs become susceptible to chromosome mis-segregation as a consequence of maternal aging. This would generate aneuploid embryos, so causing increased and birth defects (Jones and Lane, 2013). However, the actual ways in which chromosome segregation errors occur remain elusive, due to a lack of direct observations of the events as they happen. Live-cell tracking of chromosomes would be the infertility most appropriate technique to answer these questions, however with only chromosomal histone labeling, previous studies failed to follow any detailed dynamics of individual bivalents (Chiang et al., 2010; Lister et al., 2010).

In our recently published paper in Development (Yun et al., 2014), we applied a chromosome-tracking approach to examine bivalent dynamics in oocytes of aged mice during the entire period of meiotic maturation, by labeling both the bivalents and their kinetochores. By tracking, we have managed to reduce the intensity of imaging to such an extent that we can follow the movements of individual bivalents with a temporal resolution of 2 minutes continuously over a 12-15 hour time window, without any noticeable loss in rates of meiotic maturation (Movie 1). In so doing we have been able to catalogue the movements of bivalents and kinetochores in a way not previously performed, and establish the effects of maternal aging on chromosome dynamics in the first meiotic division (MI), through to metaphase II arrest (metII). Real-time tracking of bivalents in aged oocytes would be informative in the following aspects: 1) to determine if the process of bivalent congression necessary for faithful segregation is affected by age; 2) to determine the origin of single chromatids, which are commonly observed on metII eggs.

Using measurement of bivalent non-alignment when its displacement was >4 mm from the spindle equator (Lane et al., 2012), congression of all bivalents was achieved at least 3 hours ahead of anaphase onset independent of age, suggesting no gross malfunctioning of bivalent congression with age. However, we did observe more frequent weakly-attached bivalents in live aged oocytes, which had no apparent histone signal between the two sister chromatid pairs. Intriguingly, these bivalents did not undergo premature separation, but instead remained associated together all through MI. Despite the above observations in MI, the main defect with age was premature separation of dyads during metII arrest. The event was captured during imaging and occurred around 2 hours after anaphase I, as the metII spindle was assembling (Movie 2). The newly formed single chromatids oscillated about the spindle equator, presumably because they have only a single kinetochore that fails to establish simultaneous attachment to both spindle poles.

In conclusion, these data show that although considerable cohesion loss occurs during MI, its consequences are observed during meiosis II, when centromeric cohesion is needed to maintain dyad integrity, consistent with human studies that have shown a prevalence of pre-division in eggs from older women (Kuliev et al., 2011). The present work highlights that biopsy of the first polar body alone, which would have been normal in most aged oocytes here, may not be an effective screening method for aneuploidy.

References

Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M. and Lampson, M. A. (2010). Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes. Current biology : CB 20, 1522-1528.

Jones, K. T. and Lane, S. I. (2013). Molecular causes of aneuploidy in mammalian eggs. Development 140, 3719-3730.

Kuliev, A., Zlatopolsky, Z., Kirillova, I., Spivakova, J. and Cieslak Janzen, J. (2011). Meiosis errors in over 20,000 oocytes studied in the practice of preimplantation aneuploidy testing. Reproductive biomedicine online 22, 2-8.

Lane, S. I., Yun, Y. and Jones, K. T. (2012). Timing of anaphase-promoting complex activation in mouse oocytes is predicted by microtubule-kinetochore attachment but not by bivalent alignment or tension. Development 139, 1947-1955.

Lister, L. M., Kouznetsova, A., Hyslop, L. A., Kalleas, D., Pace, S. L., Barel, J. C., Nathan, A., Floros, V., Adelfalk, C., Watanabe, Y. et al. (2010). Age-related meiotic segregation errors in mammalian oocytes are preceded by depletion of cohesin and Sgo2. Current biology : CB 20, 1511-1521.

Yun, Y., Lane, S. I. and Jones, K. T. (2014). Premature dyad separation in meiosis II is the major segregation error with maternal age in mouse oocytes. Development 141, 199-208.

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